Phosphate-buffered saline-based nucleofection of primary endothelial cells

Abstract

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.

Figure 1

Transfection of primary human dermal lymphatic endothelial cells (LECs) using Lipofectamine and PEI. (A) Primary LECs were transfected with 0.5 μg of pmaxGFP that was mixed with 1.5 μg (a–c) or 0.5 μg (d–f) of Lipofectamine 2000 or with 1.5 μg (g–i) of PEI. After 48-hours, transfected cells were stained with DAPI. GFP-positive cells (a,d,g), DAPI-stained nuclei (b,e,h) and merged images (c,f,i) were shown. Bar: 100 μm. (B) Transfection efficiency of Lipofectamine or PEI was expressed by a percentage of GFP positive cells over all DAPI-positive nuclei ( > 400). Cell viability was determined by counting trypan blue-negative live cells over all cells counted ( > 500). Asterisk indicates p < 0.05 in reference to the first two conditions.

Figure 2

Nucleofection of primary human dermal lymphatic endothelial cells. (A) Primary LECs were nucleofected by using the Amaxa Lung Microvascular Endothelial Cell kit reagent (a–c), PBS (d–f) or endothelial basal media (EBM) (g–i). After 48-hours, nucleofected cells were stained with DAPI. GFP-positive cells (a,d,g), DAPI-stained nuclei (b,e,h) and merged images (c,f,i) were shown. Bar: 100 μm. (B) Transfection efficiency and cell viability for the kit reagent, PBS and EBM were expressed by a percentage of GFP positive cells and trypan blue-negative live cells, respectively. Asterisks indicate p < 0.05 in reference to the reagent condition. (C) PBS-mediated nucleofected LECs were further analyzed by flow cytometry. pmaxGFP-transfected LECs were compared against LECs transfected with a non-GFP vector (pcDNA3). About 61.3% cells showed GFP-signal above the background control level. (D) Yields of total RNA and whole cell lysate were compared for the reagent, PBS and EBM-nucleofected LECs. Asterisks indicate p < 0.05 in reference to the reagent condition. (E) Primary LECs were transfected with siRNA against Prox1 and/or COUP-TFII by using PBS-mediated nucleofection. After 48-hours, the steady-state level of Prox1 and COUP-TFII proteins were determined by western blotting analyses. siCTR, a negative control siRNA targeting the firefly luciferase; siProx1, siRNA for Prox1; siCOU, siRNA for COUP-TFII; siBoth, mixture of siRNAs for Prox1 and COUP-TFII.

Figure 3

PBS-based nucleofection of primary human dermal blood vascular endothelial cells (BECs), primary HUVECs and L6 rat myoblasts. Primary BECs (A,B), HUAECs (C,D) and L6 rat myoblasts were nucleofected with 1 μg of pmaxGFP and after 48-hours, images of GFP-transfected cells (A,C,E), DAPI-stained nuclei (B) and bright field (D,F) of the cells were captured. Bars: 100 μm. (G) Transfection efficiency and cell viability of PBS-based nucleofected cells were expressed by a percentage of GFP positive cells and trypan blue-negative live cells by counting > 400 cells. (H,I) Flow cytometry analyses of PBS-based nucleofected HUAECs with 1 μg (H) and 4 μg (I) of pmaxGFP.

Figure 4

Determination of the optimal conditions of PBS-based nucleofection for human thyroid cancer cells (TPC1). Human thyroid cancer cells TPC1 were nucleofected with 2 μg of pmaxGFP using Solution V, Solution L or PBS and different nucleofection programs (A-20, T-20, T-30, X-01, X-05, L-29 and D-23). After 48-hours of nucleofection, nucleofection efficiency (A) and cell viability (B) of each condition were measured. Images of GFP-transfected cells (C) and bright field (D) of TPC1 nucleofected using PBS and the X-01 program were shown. Bars: 100 μm.